Kidney-targeted drug delivery systems

ABSTRACT

A composition including an elastin-like polypeptide (ELP) coupled to a kidney targeting peptide and. a therapeutic agent is provided. A method of delivering a therapeutic agent to a subject in need thereof comprising: administering to the subject an effective amount of a composition comprising: an elastin-like polypeptide (ELP), a kidney targeting agent coupled to the ELP and a targeting agent and/or a drug binding domain coupled to the ELP, wherein the ELP includes an amino acid sequence having at least about repeats of the amino acid sequence GVPGX (SEQ ID NO: 1), and wherein the composition enhances the deposition and retention of the therapeutic agent in the kidney relative to the non-conjugated therapeutic.

RELATED APPLICATIONS

This application claims priority from International Patent ApplicationNo. PCT/US2015/060438, filed Nov. 12, 2015, which claims priority fromU.S. Provisional Application Ser. No. 62/078,752 filed Nov. 12, 2014,the entire disclosures of which are incorporated herein by thisreference.

STATEMENT OF GOVERNMENT SUPPORT

This presently-disclosed subject matter was made with government supportunder grant number R01HL095638 and R01HL121527 awarded by the NationalInstitutes of Health. The government has certain rights in it.

TECHNICAL FIELD

The presently-disclosed subject matter generally relates to acomposition and method for therapeutic agent delivery of kidney diseasestreatment. More particularly, the presently-disclosed subject matterrelates to a composition comprising an elastin-like polypeptide (ELP)coupled to kidney targeting peptides and a therapeutic agent or agents,and a method of delivering the composition to a subject in need thereof.

INTRODUCTION

The kidney plays a critical role in sodium/water balance, maintenance ofblood pressure, and removal of waste products from the circulatorysystem. Damage or disease to the kidneys can have very seriousconsequences including the often irreversible need to place patients onhemodialysis for the remainder of their life. Therefore, the kidney isan important drug target, and therapies that can prevent loss of kidneyfunction or even restore function in damaged kidneys would have greatclinical value.

Chronic kidney disease (CKD) is a progressive disorder affecting almost14% of the general adult population, and this disease has shown acontinuous growth over the past 2 decades. Patients with CKD have higherrates of hospitalization, greater mortality, shorter life expectancy,and their healthcare costs are up to 5 times more expensive than non-CKDpatients. Thus, treatments to slow, halt, or reverse the progression ofCKD could have a significant impact. Chronic renal vascular disease(RVD), often associated with renal artery stenosis, can deterioraterenal function and lead to CKD and end-stage renal disease. Despite theavailability of treatments for RVD including drugs and renalangioplasty, renal function does not improve or even deteriorates inover half of the patients undergoing these treatments. This evidenceshows that treatments available are still largely ineffective andhighlights a pressing need for novel therapeutic strategies for thegrowing population of patients suffering from RVD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a series of images and a graph depicting thebiodistribution of kidney targeted ELPs in the rat. Rats were givenfluorescently labeled ELP, Synb1-ELP, Tat-ELP, and KTP-ELP byintravenous injection. Whole-body fluorescence was determined by in vivofluorescence imaging at various times after injection (A). The meanfluorescence intensity was determined at each time point and plotted toshow tissue deposition and clearance (B). N=3 rats per treatment group.

FIG. 2 includes a series of images and graphs demonstrating theenhancement of kidney specificity using Kidney Targeting Peptides. Ratswere administered fluorescently labeled ELP, SynB1-ELP, Tat-ELP, orKTP-ELP, and organ biodistribution was determined by ex vivofluorescence imaging (A.). Quantitative analysis showed that the highestaccumulation of all peptides was in the kidney, and the targeting agentssignificantly increased kidney deposition (B.). KTP-ELP had the highestspecificity for the kidney as assessed by kidney:liver and kidney:heartratios (C. and D.).

FIG. 3 includes a series of slide scans and micrographs depicting theintrarenal distribution of renally targeted ELPs. Four hours afterintravenous infusion of fluorescently labeled ELP, SynB1-ELP, Tat-ELP,or KTP-ELP, the kidneys were rapidly frozen and cut into 20 μm sections.A. Slides were scanned using a fluorescence slide scanner. Identicalscan settings were used in order to directly compare the total kidneylevels. B. Slides were stained with several vascular markers and imagedusing a fluorescence microscope and 20× objective. Shown is theintrarenal distribution of KTP-ELP. Tat-ELP and SynB1-ELP had similarintrarenal distribution (not shown).

FIG. 4 includes fluorescence photographs and a bar graph showing thatKTP enhances ELP deposition in the swine kidney after IV administration.Pigs (n=3 per agent) were given fluorescently labeled ELP or KTP-ELP byintravenous injection. Organ distribution was determined 4 h afterinjection by er vivo whole organ fluorescence imaging (A) and quantifiedrelative to standard curves of each agent (B).

FIG. 5 includes a series of bar graphs demonstrating that KTP-ELPenhances ELP binding to human renal cells. Cell binding (A-C) and cellsurvival (D-F) of SynB1-ELP, Tat-ELP, and KTP-ELP relative to ELPcontrol were determined by flow cytometry and a cell viability assay,respectively, in primary human glomerular microvascular endothelialcells (A,D), primary human podocytes (B,E), and primary human proximaltubule epithelial cells (C,F).

FIG. 6 contains a scatter plot and tabular data depicting thepharmacokinetics of intrarenal ELP-VEGF in the Pig. Three pigs (averageweight 49.2 kg) were given fluorescently labeled ELP-VEGF by directintrarenal administration. A balloon was inflated o block blood flowinto and out of the injected kidney for three minutes. The balloon wasreleased, and plasma was sampled to determine ELP-VEGF levels. Plasmalevels were determined by direct detection of fluorescence and fit to atwo compartment pharmacokinetic model.

FIG. 7 includes a series of images and a graph depicting the intrarenalbiodistribution of ELP-VEGF in the Pig. Three pigs were givenfluorescently labeled ELP-VEGF by direct intrarenal administration.Organ distribution was determined 4 h after injection by ex vivo wholeorgan fluorescence imaging.

FIG. 8 includes a series of bar graphs and images demonstrating thatELP-VEGF maintains its pro-angiogenic activity. A. Stimulation of HGMEcell proliferation was determined by exposing HGME cells to ELP control,unconjugated VEGF, or ELP-VEGF for 72 h, and viable cells were detectedusing the MTS cell proliferation assay. B. and C. To determine ifELP-VEGF could stimulate tube formation in primary endothelial cells,HGME cells were plated on growth factor reduced Matrigel, and the mediawas supplemented with the indicated proteins. Tube formation wasassessed after 5 h of exposure to the proteins. D. and E. To determinewhether ELP-VEGF functions as a chemokine for primary endothelial cells,HGME cells were plated in the top well of Matrigel-coated Boydenchambers, and media in the bottom well was supplemented with the testproteins. Migrating cells were detected on the bottom surface of themembranes by crystal violet staining after 16-24 h of protein exposure.*Levels are significantly higher than untreated cells as assessed by aone-way ANOVA and post-hoc Bonferroni multiple comparison.

FIG. 9 includes a series of graphs and images demonstrating the effectof intrarenal ELP-VEGF on renal function in RVD. FIG. 9A shows thatintra-renal administration of ELP-VEGF improved renal function, corticaland medullary vascular density in the stenotic kidney. Effect ofintra-renal ELP-VEGF on renal function (top) and microvascular (MV)architecture (3D micro-CT reconstruction, bottom) and quantification innormal, renovascular disease (RVD), RVD+ELP, and RVD+ELP-VEGF treatedkidneys. *p<0.05 vs. Normal; †p<0.05 vs. RVD/RVD+ELP; ‡p<0.05 vs. 6weeks. FIG. 9B shows that intra-renal administration of ELP-VEGFimproved the vascular density of both small and larger MV diameters inthe cortex and medulla of the stenotic kidney. Cortical and medullaryquantification of microvascular (MV) density divided by MV diameter innormal, renovascular disease (RVD), and RVD+ELP-VEGF treated kidneys.*p<0.05 vs. Normal; †p<0.05 vs. RVD.

FIG. 10 includes bar graphs and tabular data demonstrating that ELP-VEGFis superior to unmodified VEGF at restoring renal function andmicrovascular density in the swine model of renal artery stenosis.Comparisons between intra-renal unbound VEGF vs. ELP-VEGF therapy on:Top—basal stenotic kidney RBF and GFR, expressed as % change compared topre-treatment values; Middle—RBF and GFR responses to intra-renalinfusion of acetylcholine; Bottom—effects of the treatments on corticaland medullary MV density (3D micro-CT reconstruction, divided by MVdiameter) in renovascular disease (RVD)+VEGF and RVD+ELP-VEGF treatedkidneys. †p<0.05 vs. RVD/RVD+VEGF; ‡p<0.05 vs. 6 weeks; ‡‡p<0.05 vs.baseline; #p=0.09 vs. RVD.

FIG. 11 includes Western blot data and bar graphs demonstrating thatIntra-renal administration of ELP-VEGF improved the expression ofangiogenic factors and promoters of mobilization and homing ofprogenitor cells in the stenotic kidney. Representative renal proteinexpression (top, 2 bands per group) of vascular endothelial growthfactor (VEGF), its receptor Flk-1, phosphorylated (p)-akt, angiopoietin(Ang)-1 and -2, Tie-, angiostatin (angio), stromal-derived factor(SDF)-1 and its receptor CXCR4, and quantification (bottom) in normal,renovascular disease (RVD), and RVD+ELP-VEGF treated kidneys. *p<0.05vs. Normal; †p<0.05 vs. RVD.

FIG. 12 includes Western blot and histological data showing thatintra-renal administration of ELP-VEGF reduced renal fibrogenic activityand attenuated podocyte damage and fibrosis in the stenotic kidney. Top:Representative renal protein expression (2 bands per group) andquantification of transforming growth factor (TGF)-β, smads-4-7, matrixmetalloproteinases (MMP)-2 and its inhibitor TIMP-1 in normal,renovascular disease (RVD), and RVD+ELP-VEGF treated kidneys. Middle:Representative pictures (from stenotic kidneys) of the glomeruli (×40),showed as examples to illustrate podocin immunoreactivity (blackarrows). Bottom: Representative trichrome pictures (from stenotickidneys) of the glomeruli and tubules, and tubule-interstitial regions(×20, showed as examples to illustrate renal damage). *p<0.05 vs.Normal; †p<0.05 vs. RVD.

FIG. 13 includes and image and a graph showing the effect of IV ELP-VEGFon renal function in RVD. A. Stenotic vs. contralateral kidney after 10weeks of RVD (4 weeks after IV ELP-VEGF). B. Change in RBF and GFR afterIV ELP-VEGF. C. Micro-CT quantification of renal MV density after IVELP-VEGF. Intra-venous ELP-VEGF improved renal function and MV densityof the stenotic kidney. *p<0.05 vs. Normal; †p<0.05 vs. RVD.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. Further, while the terms used herein are believed to bewell-understood by one of ordinary skill in the art, definitions are setforth to facilitate explanation of the presently-disclosed subjectmatter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

The presently-disclosed subject matter relates to a composition andmethod for therapeutic agent delivery for treatment of kidney diseases.More particularly, the presently-disclosed subject matter relates to acomposition comprising an elastin-like polypeptide (ELP) coupled to akidney targeting agent and a therapeutic agent or agents, and a methodof delivering the composition to a subject in need thereof.

As used herein, the term “elastin-like polypeptide” or “ELP” refers to asynthetic protein containing structural peptide units, which may berepeating units, structurally related to, or derived from, sequences ofthe elastin protein. ELP is a macromolecular carrier that has severaladvantages. It is an inert and biodegradable macromolecule, giving it agood pharmacokinetic profile and very low immunogenicity. Also, asopposed to chemically synthesized polymers, ELP is expressed in andeasily purified from E. coli. Further, the sequence of a particular ELPcan be controlled such that it is possible to generate chimeras of ELPfused to therapeutic proteins or peptides or to add reactive sites forattachment of therapeutic agents. Such ELP chimeras provide certaintherapeutic advantages to the therapeutic agent, such as comparativelybetter stability, solubility, bioavailability, half-life, persistence,and/or biological action of the therapeutic proteinaceous component orattached small molecule drug.

In some embodiments, the presently-disclosed subject matter provides akidney targeted drug delivery system composed of a biopolymer carriermodified with a kidney targeting agent and a drug binding domain or adirectly fused therapeutic peptide or protein. The kidney targeted drugcarrier consists of one of several targeting peptides that conferkidney-specific delivery fused to a biopolymer based on elastin-likepolypeptide (ELP) [1-3]. In some embodiments, the ELP domain consists ofrepeating units of the GVPGX motif, in which X can be any amino acidexcept proline. In some embodiments, a drug binding domain and/or atherapeutic peptide or protein is also fused to the ELP biopolymer. Insome embodiments, the drug binding domain consists of a regioncontaining multiple cysteine or lysine residues that can be used forcovalent attachment of drugs. In some embodiments, in addition tocovalent drug attachment, the therapeutic domain might containtherapeutic peptides or proteins designed to intervene in diseaseprocesses of the kidney.

When all domains are included in the same molecule, the targeting domainincreases kidney deposition and confers kidney specificity, the ELPbiopolymer provides mass that confers protection from degradation andrapid renal clearance, and the therapeutic domain and/or drug bindingdomain provides a mechanism for intervening in disease processes of thekidney. ELPs can be fused to virtually any therapeutic compound bysimple molecular biology techniques. Thus, determining the feasibilityof using ELP technology for renal therapy could have clinicalramifications that go beyond chronic RVD and may extend to CKD fromdifferent etiologies.

In some embodiments, the presently disclosed subject matter provides acomposition comprising an elastin-like polypeptide (ELP), a kidneytargeting agent coupled to the ELP, and a therapeutic agent and/or adrug binding domain coupled to the ELP. In some embodiments, thepresently disclosed subject matter further includes a pharmaceuticallyacceptable carrier. In some embodiments, the ELP includes an amino acidsequence having at least about 5 repeats of the amino acid sequenceGVPGX (SEQ ID NO: 1), and the composition enhances the deposition andretention of the therapeutic agent in the kidney relative to thenon-conjugated therapeutic. In some embodiments, the ELP includes anamino acid sequence comprising about 5 repeats to about 320 repeats ofthe amino acid sequence GVPGX. In some embodiments, X in the sequenceGVPGX is any amino acid except proline. In some embodiments, X in theamino acid sequence GVPGX is Val, Ala, and Gly in a ratio range of about0-1:0-8:0-8.

The terms “polypeptide”, “protein”, and “peptide”, which are usedinterchangeably herein, refer to a polymer of the protein amino acids,or amino acid analogs, regardless of its size or function. Although“protein” is often used in reference to relatively large polypeptides,and “peptide” is often used in reference to small polypeptides, usage ofthese terms in the art overlaps and varies. The term “polypeptide” asused herein refers to peptides, polypeptides, and proteins, unlessotherwise noted. The terms “protein”, “polypeptide”, and “peptide” areused interchangeably herein when referring to a gene product. Thus,exemplary polypeptides include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, and analogs of the foregoing.

The term “kidney targeting agents” refers to short peptides designed tohave specificity for the vascular beds or other cell types of specificorgans such as kidney.

The term “therapeutic agent” and the like is used herein to refer tosubstances that can alter, inhibit, activate, catalyze, or otherwiseaffect a biological or chemical event in a subject. In some embodimentsa therapeutic agent has the effect of treating a disease, condition, ordisorder in a subject, and possibly in the kidney of a subject.Exemplary active agents include, but are not limited to, enzymes,organic catalysts, ribozymes, organometallics, proteins, glycoproteins,peptides, polyamino acids, antibodies, nucleic acids, steroidalmolecules, antibiotics, antibacterial agents, anti-inflammatory agents,antivirals, antimycotics, anticancer agents, analgesic agents,antirejection agents, immunosuppressants, cytokines, carbohydrates,oleophobics, lipids, pharmaceuticals (i.e., drugs; including smallmolecules), chemotherapeutics, and combinations thereof.

Non-limiting examples of the ELP sequences include an amino acidsequence in which X in the GVPGX sequence is Val, Ala, and Gly in a1:8:7 ratio (SEQ ID NO: 2), Gly (SEQ ID NO: 3), Val, Ala, and Gly in a1:4:3 ratio (SEQ ID NO: 4), or a combination thereof.

Additionally, non-limiting examples of the kidney targeting agentsinclude a kidney targeting peptide (SEQ ID NO: 5), a kidney targetingpeptide (SEQ ID NO: 6), a Tat peptide (SEQ ID NO: 7), a SynB1 peptide(SEQ ID NO: 8), or a combination thereof.

Moreover, non-limiting examples of the drug binding domain includesrepeats of the sequence GGC (SEQ ID NO: 9), the sequence GC (SEQ ID NO:10), the sequence GGK (SEQ ID NO: 11), and the sequence GK (SEQ ID NO:12).

Further provided in some embodiments of the presently disclosed subjectmatter, is a therapeutic agent includes at least one growth factor. Insome embodiments, the growth factor includes VEGF, HGF, b-FGF, TGF-β,and HIF. In some embodiments, the therapeutic agent includes a VEGFselected from VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E, and P1GF.

The term “pharmaceutically acceptable carrier” refers to sterile aqueousor nonaqueous solutions, dispersions, suspensions or emulsions, as wellas sterile powders for reconstitution into sterile solutions ordispersions just prior to use. These compositions can also containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. Prevention of the action of microorganisms can beensured by the inclusion of various antibacterial and antifungal agentssuch as paraben, chlorobutanol, phenol, sorbic acid and the like. It canalso be desirable to include isotonic agents such as sugars, sodiumchloride and the like. Depending upon the ratio of drug to polymer andthe nature of the particular polymer employed, the rate of drug releasecan be controlled. The formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use.

Further, in some embodiments, the presently-disclosed subject matterprovides a method of delivering a therapeutic agent to a subject in needthereof. The method includes administering to the subject an effectiveamount of a composition. The composition includes an elastin-likepolypeptide (ELP), a kidney targeting agent coupled to the ELP, and atherapeutic agent and/or a drug binding domain coupled to the ELP. Insome embodiments, the ELP includes an amino acid sequence having atleast about 5 repeats of the amino acid sequence GVPGX (SEQ ID NO: 1),and the composition enhances the deposition and retention of thetherapeutic agent in the kidney relative to the non-conjugatedtherapeutic. In some embodiments, the ELP includes an amino acidsequence comprising about 5 repeats to about 320 repeats of the aminoacid sequence GVPGX, and X in the sequence GVPGX is any amino acidexcept proline. In some embodiments, the X in the amino acid sequenceGVPGX is Val, Ala, and Gly in a ratio range of about 0-1:0-8:0-8. Insome embodiments, the ELP includes the amino acid sequence GVPGX, and Xis Val, Ala, and Gly in a 1:8:7 ratio (SEQ ID NO: 2) repeated between 5and 320 times. In some embodiments, the ELP comprises the amino acidsequence GVPGX, and wherein X is Gly (SEQ ID NO: 3) repeated between 5and 320 times. In some embodiments, the ELP comprises the amino acidsequence GVPGX, and X is Val, Ala, and Gly in a 1:4:3 ratio (SEQ ID NO:4) repeated between 5 and 320 times.

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired result or to have an effect on anundesired condition. For example, a “therapeutically effective amount”refers to an amount that is sufficient to achieve the desiredtherapeutic result or to have an effect on undesired symptoms, but isgenerally insufficient to cause adverse side affects. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts.

Further provided, in some embodiments of the presently disclosed subjectmatter, is a targeting agent that is used to increase kidney depositionand specificity. The incorporation of the targeting agent increaseskidney deposition and specificity of the delivered therapeutic agentand/or a drug binding domain. In some embodiments, the kidney targetingagent is a kidney targeting peptide having SEQ ID NO: 5. In someembodiments, the kidney targeting agent is a peptide having SEQ ID NO:6. In some embodiments, the kidney targeting agent is a Tat peptidehaving SEQ ID NO: 7. In some embodiments, the kidney targeting agent isa SynB1 peptide having SEQ ID NO: 8. In some embodiments, non-limitingexamples of the drug binding domain includes repeats of the sequence GGC(SEQ ID NO: 9), repeats of the sequence GC (SEQ ID NO: 10), repeats ofthe sequence GGK (SEQ ID NO: 11), and repeats of the sequence GK (SEQ IDNO: 12). In some embodiments, the therapeutic agent includes at leastone growth factor selected from the group consisting of VEGF, HGF,b-FGF, TGF-β, and HIF. Non-limiting examples of VEGF include VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, andP1GF. In some embodiments, the composition is administeredintra-renally, intravenously, intraperitoneally, orally, intranasally,or subcutaneously.

In this regard, the term “administer” refers to any method of providinga compound or composition thereof to a subject. In some embodiments,suitable methods for administering a therapeutic composition inaccordance with the methods of the presently-disclosed subject mattermay include, but are not limited to, intra-renal administration,intravenous administration, intraperitoneal administration, oraladministration, intranasal administration, subcutaneous administration,systemic administration, parenteral administration (includingintravascular, intramuscular, and/or intraarterial administration),buccal delivery, rectal delivery, inhalation, intratrachealinstallation, surgical implantation, transdermal delivery, localinjection, and hyper-velocity injection/bombardment.

Further provided, in some embodiments of the presently-disclosed subjectmatter are methods for the treatment of various diseases and disordersusing the exemplary ELP-therapeutic agent-containing compositionsdescribed herein. In some embodiments, the presently-disclosed subjectmatter includes a method of treating a kidney disease or disorder in asubject wherein the subject is administered an effective amount of acomposition comprising an ELP coupled to a kidney targeting agent and atherapeutic agent and/or a drug binding protein. In some embodiments,the ELP includes an amino acid sequence having at least about 5 repeatsof the amino acid sequence GVPGX (SEQ ID NO: 1), and the compositionenhances the deposition and retention of the therapeutic agent in thekidney relative to the non-conjugated therapeutic. Exemplary diseases ordisorders that can be treated in accordance with the presently-disclosedsubject matter include, but are not limited to, Chronic kidney disease(CKD), Chronic renal vascular disease (RVD), end-stage renal disease.

In some embodiments the method for administering the present compoundsand compositions further include treating a disease or condition in thesubject. The terms “treatment” or “treating” refer to the medicalmanagement of a patient with the intent to cure, ameliorate, stabilize,or prevent a disease, pathological condition, or disorder. This termincludes active treatment, that is, treatment directed specificallytoward the improvement of a disease, pathological condition, ordisorder, and also includes causal treatment, that is, treatmentdirected toward removal of the cause of the associated disease,pathological condition, or disorder. In addition, this term includespalliative treatment, that is, treatment designed for the relief ofsymptoms rather than the curing of the disease, pathological condition,or disorder; preventative treatment, that is, treatment directed tominimizing or partially or completely inhibiting the development of theassociated disease, pathological condition, or disorder; and supportivetreatment, that is, treatment employed to supplement another specifictherapy directed toward the improvement of the associated disease,pathological condition, or disorder.

Furthermore, the term “subject” is inclusive of both human and animalsubjects. Thus, veterinary uses are provided in accordance with thepresently disclosed subject matter and the presently-disclosed subjectmatter provides methods for preventing oxidative damage in mammals suchas humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. The following examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the present invention.

EXAMPLES Example 1 Targeting Peptides Increase Total Renal Depositionand Enhance the Renal Specificity of ELP Biodistribution

A biodistribution study was performed to determine whether increasingELP levels in the kidneys using targeting peptides was possible. The ELPmolecule was fused to one of two cell penetrating peptides (CPPs) (SynB1and Tat [4, 5] or to a peptide found to have specificity for the kidney(Kidney Targeting Peptide. KTP [6]). Each polypeptide was labeled with afluorophor and administered by IV injection at a dose of 100 mg/kg inhairless Sprague Dawley rats. The whole-body fluorescence of the animalsthroughout the experiment is measured by in vivo imaging. As shown inFIG. 1, when the untargeted ELP carrier was injected, the fluorescencespread throughout the body and reached a peak intensity approximately 1hour after the injection, then the fluorescence level slowly decreased.In contrast, the SynB1-ELP and the KTP-ELP polypeptides achieved muchhigher levels throughout the body, and the levels were just beginning topeak four hours after the injection. These data reveal that the use ofthe targeting peptides increases extravasation and tissue uptake of thedrug carrier and therefore slows its clearance from the body tissues.

The whole-body in vivo fluorescence provides a measurement of totaltissue polypeptide levels, but it cannot resolve the individual organbiodistribution. In order to measure the biodistribution, the majororgans were removed, and polypeptide levels were determined byquantitative whole organ ex vivo imaging four hours after the injection.As shown in FIG. 2, the unmodified ELP accumulated most highly in thekidney and the liver. When the ELP was modified with the cellpenetrating peptides or the KTP, the kidney levels increaseddramatically (over five-fold, FIG. 2B). Both cell penetrating peptidesand the KTP achieved similar kidney levels. However, when kidneyspecificity was assessed by measuring the kidney:liver and thekidney:heart ratios, the KTP proved to be by far the most specific(FIGS. 2C and D). In fact, KTP-ELP accumulated in the kidneys at levels15-fold higher than in the liver and as much as 150-fold higher than inother organs including the heart, brain, and spleen. These datademonstrate that KTP is an effective targeting agent for increasing bothtotal renal accumulation and renal specificity of the ELP drug carrier,and they are the first demonstration of the use of targeting peptides toachieve kidney-specific delivery of the ELP carrier.

In addition to the whole-organ ex vivo imaging, the kidneys were frozenand sectioned to determine the intrarenal distribution of thepolypeptides. Fluorescence slide scanning revealed that all polypeptideswere mostly confined to the renal cortex (FIG. 3A). Also, consistentwith the whole-organ imaging, SynB1-ELP, Tat-ELP, and KTP-ELPaccumulated to very high levels relative to the untargeted ELPbiopolymer. When examined microscopically (FIG. 3B), KTP-ELP waslocalized around the nephron (marked by synaptopodin staining) and wasdetectable in both the blood vessel walls (as indicated by CD31staining) and in the surrounding proximal and distal tubules. Imagestaken in the outer medulla and co-stained with calbindin to mark thecollecting ducts also showed high levels of KTP-ELP within the ductalepithelial cells. SynB1-ELP and Tat-ELP had very similar intrarenaldistributions (data not shown).

To insure that the ability of KTP to target ELP to the kidneys was notspecific to rats, a similar experiment was conducted in swine. Domesticcrossbred female pre-juvenile pigs (sus scrofa domestica) wereadminstered ELP or KTP-ELP (n=3 pigs/agent) by IV injection. Ex vivoquantitative fluorescence histology was performed as described above. Asshown in FIG. 4, KTP-ELP accumulated in the swine kidney at levels 5.4fold higher than the untargeted ELP, and kidney KTP-ELP levels were4.8-fold higher than liver, almost two-fold higher than lung, and over50-fold higher than heart and spleen. These data demonstrate that KTP iseffective for kidney targeting in a predictive preclinical model and isnot species specific.

We also sought to determine if KTP could enhance ELP binding to primaryhuman renal cells and to identify which cell type KTP has affinity for.Primary human glomerular microvascular endothelial cells (HOME), primaryhuman podocytes, and primary human renal proximal tubule epithelialcells (HRPTEpC) were cultured in vitro and exposed to 10 μM ELP orKTP-ELP. Cells were also exposed to the cell penetrating peptide—fusedELPs SynB1-ELP and Tat-ELP as comparators. As shown in FIG. 5. KTPenhanced ELP binding to all renal cell types. Binding was increased 3fold in HGME, 1.7 fold in podocytes, and 3 fold in HRPTEpC by KTP-ELPrelative to ELP control. This was in contrast to the CPP-fused ELPs. Tatonly enhanced ELP binding to HRPTEpC and actually reduced binding topodocytes. SynB1 only enhanced ELP binding to HGME cells and alsoreduced binding to podocytes (FIG. 5A). We also tested whether KTP-ELPhad any toxicity to the human cell lines. Each cell line was incubatedwith ELP, SynB1-ELP, Tat-ELP, or KTP-ELP at concentrations up to 40 μMfor 72 h, and cell number was determined using the MTS assay. KTP showedno toxicity to any cell line tested, and it even stimulatedproliferation of HRPTEpC (FIG. 5B). In contrast, Tat-ELP was toxic HGMEcells. SynB1-ELP showed no cytotoxicity. These data demonstrate that KTPhas affinity for several renal cell lines, and, in contrast to CPPs, itincreases ELP binding to all renal cell types tested.

Example 2 ELP-Fused Vascular Endothelial Growth Factor (VEGF) Depositsin the Kidney and Improves Renal Function in a Swine Model ofRenovascular Disease

Chronic kidney disease (CKD) is a progressive disorder affecting almost14% of the general population, and the prevalence of this disease hascontinuously grown over the past 2 decades [7]. CKD is an independentrisk factor for cardiovascular morbidity and mortality, as patients withdiagnosed cardiovascular disease show a staggering 40.8% prevalence ofCKD, a number that has doubled in less than 20 years [7]. Patients withCKD have higher rates of hospitalization, greater mortality, shorterlife expectancy, and their healthcare costs are up to 5 times moreexpensive than non-CKD patients, which represent an enormous burden tothe healthcare budget. Thus, treatments to slow, halt, or reverse theprogression of CKD could have a significant impact.

Chronic RVD can deteriorate renal function and lead to CKD and end-stagerenal disease. It affects between 9-11% of the general population, butthis number goes up in patients with diagnosed coronary artery orperipheral vascular disease (about 30%), and are much higher in olderpatients (up to 60% in patients>65 years) [8-10]. The main cause of RVDis renal artery stenosis, often due to atherosclerosis. Although thevascular obstruction is the initial and possibly main instigator ofrenal injury, therapeutic strategies that aim to resolve the vascularstenosis such as renal angioplasty and stenting are effective inrecovering renal function in less than half of the cases. The disparitybetween technical success and outcomes has served as the impetus fornumerous trials to assess the efficacy of medical therapy vs.interventions in this disease, focusing in two major end points:reduction of hypertension and recovery of renal function. Nevertheless,the outcomes of RVD are still poor. Although numerous trials have beencritiqued because of flaws in design and follow up, the results weighedmore towards the conclusion that there are no major benefits achieved byrenal angioplasty compared to medical treatment that would justify therisk of revascularization procedures [11]. Consequently, there is anoticeable lack of consensus regarding the best therapeutic strategy forthese patients. Hence, more effective treatments are needed and thetechnology described within represents a new therapeutic strategy thathas not been previously tested for renal therapy.

Damage of the small vessels in the kidney is a common pathologicalfeature in CKD and end stage renal disease irrespective of the cause.Furthermore, major cardiovascular factors and causes of CKD such ashypertension or diabetes have been shown to associate with intra-renalmicrovascular (MV) rarefaction that is observed before deterioration ofrenal function. These support the notion of a potential cause-effectrelationship and suggest a pathophysiological role of MV damage on theprogression of renal dysfunction. Over the past 14 years, a unique swinemodel of RVD was developed that mimics the progressive nature of renalinjury, hypertension, and cardiovascular risk found in humans with RVD.Moreover, physiological imaging techniques were developed and validatedusing high-resolution computerized tomography (CT) to measure renalregional volumes, total renal blood flow (RBF), glomerular filtrationrate (GFR), tubular dynamics, and endothelial function; and micro-CT tostudy the 3D architecture of the renal microcirculation in situ. Thesetechniques allow us to non-invasively and serially follow the timecourse of the deterioration of the kidney in an integrative fashion andwith previously unavailable accuracy. Progressive loss of renal functionand tissue damage in RVD is accompanied by marked and progressive renalmicrovascular damage and loss in the stenotic kidney (evident in bothrenal cortex and medulla), which is mediated by a progressive decreasein renal expression and availability of VEGF and a defective renalangiogenesis and vascular repair [12-15].

ELP-VEGF is retained in the kidney after intrarenal administration inthe pig. To determine if the ELP-delivered VEGF could be retained in thekidney in the swine RVD model, a biodistribution study in the pig wasconducted. Three pigs (average weight 49.2 kg) were administeredfluorescently labeled ELP-VEGF by direct intrarenal injection underfluoroscopy guidance. A balloon catheter was inflated for three minutesfollowing the injection, then the balloon was deflated to allow blood tocirculate through the kidney. Blood was sampled from the jugular vein atfixed time-points, and plasma fluorescence measurements were taken tomonitor ELP-VEGF levels. The distribution phase half-life was 2.95minutes and the terminal plasma half-life was 810.1 minutes (FIG. 6).Four hours after injection, the pigs were sacrificed and the organsanalyzed by ex vivo fluorescence imaging. As shown in FIG. 7, theinjected kidney retained the ELP-VEGF, showing tissue levels nearlythree fold higher than the contralateral kidney or any other organ. Someprotein did enter systemic circulation, as evidenced by its detection inthe contralateral kidney and liver. However, these results demonstratethat intrarenal administration is a viable route for delivery ofELP-VEGF, and kidney levels will be increased further when the ELP-VEGFis fused to the KTP.

ELP-VEGF is equally as active as free vegf in primary human glomerularmicrovascular endothelial (hgme) cells. Primary Human GlomerularMicrovascular Endothelial (HGME) cells were used to insure the signalingproperties of VEGF were retained even after fusion to the ELP carrier.As shown in FIG. 8A, both unbound VEGF and ELP-VEGF stimulatedproliferation of HOME cells, while the ELP polypeptide alone had noeffect on HGME proliferation. Furthermore, no significant differenceswere seen in the potency of the unbound cytokine and the ELP-fused VEGF,suggesting that the ELP-fused VEGF is still able to bind its receptor.To test this further, HGME cells were used in to a tube formation assayon growth factor reduced Matrigel. As shown in FIG. 8B, very little tubeformation was observed on this matrix without additional stimulation.However, when the media was supplemented with unbound VEGF or ELP-VEGF,tube formation was significantly induced. Quantification of tubes pervisual field showed that both unbound VEGF and ELP-VEGF significantlyinduced tube formation relative to untreated cells (FIG. 8C). There werealso more average tubes per field in the ELP control-treated samples,though the difference did not reach statistical significance. Finally,to assess the ability of ELP-VEGF to serve as a chemokine for HGMEcells, a Matrigel migration assay was used. As shown in FIG. 8D andquantified in FIG. 8E, both unbound VEGF and ELP-VEGF strongly inducedHGME cell migration through Matrigel, while the control ELP had noeffect. Again, there was no difference in potency between VEGF andELP-VEGF.

Single-dose intra-renal elp-vegf causes improvement in renal function ina swine model of chronic rvd. To determine whether administration ofELP-VEGF into the stenotic kidney has an impact on renal function andmicrovascular architecture, 7 pigs were treated after 6 weeks of RVDwith a single infusion of ELP-VEGF (100 μg/kg). An additional 7 pigswith RVD received placebo and were used as controls. Single-kidneyfunction was quantified in vivo in all pigs before and 4 weeks aftertreatments/placebo. Pigs were observed for a total of 10 weeks and theneuthanized. Kidneys were then removed and micro CT studies (to quantifythe impact of ELP/placebo on the renal microvasculature) and proteinexpression studies performed. It was observed that administration ofELP-VEGF significantly improved renal function compared to placebo (FIG.9A, top). The improvement was specific to ELP-VEGF, as the ELP controlpolypeptide had no effect on renal function. The stenotic kidney showeda significant reduction in cortical and medullary microvascular densityaccompanied by substantial microvascular remodeling compared to normalcontrols (FIG. 9A, bottom). Notably, intra-renal ELP-VEGF significantlyimproved both cortical and medullary microvascular density andremodeling of small and large microvessels (0-500 μm in diameter), whichwas evident throughout the renal parenchyma (FIG. 9B).

ELP-VEGF is more effective than unconjugated vegf for improvement ofrenal function. A single intra-renal administration of free VEGF₁₂₁significantly improved stenotic RBF but not GFR (p<0.05 and p=NS,respectively, vs. pre-treatment values) and the magnitude of thosechanges was significantly less compared to ELP-VEGF therapy (FIG. 10,top). Furthermore, intra-stenotic kidney infusion of acetylcholine(quantified at 10 weeks) improved RBF but not GFR in free VEGF treatedkidneys whereas both RBF and GFR were improved in after ELP-VEGF (FIG.10, middle). Finally, free VEGF therapy improved MV density only inthose cortical microvessels under 200 μm in diameter and not in largermicrovessels (200-500 μm in diameter, FIG. 10, bottom. Overall, thesefindings strongly support a superior efficacy of ELP-VEGF therapy overunconjugated VEGF.

ELP-VEGF activates VEGF signaling in the stenotic kidney. Kidneys fromthe efficacy study were examined by Western blot to confirm activationof VEGF signaling at the experimental endpoint. Expression of VEGF, thereceptor Flk-1, angiopoietin (Ang)-1 and -2 and the Tie-2 receptor weresignificantly reduced in RVD but largely restored and accompanied byimproved expression of phosphorylated (p)-akt, stromal-derived factor(SDF)-1 and the CXCR4 receptor, and attenuated expression ofanti-angiogenic angiostatin (angio) after ELP-VEGF therapy, suggesting apro-angiogenic milieu in the stenotic kidney of ELP-VEGF treated pigs(FIG. 11).

ELP-VEGF reduces inflammatory activity and fibrotic damage in thestenotic kidney. ELP-VEGF therapy decreased the renal concentration oftumor necrosis factor (TNF)-α (Untreated pigs 9.8±1.4 pg/mg tissue; RVD19.4±0.6 pg/mg tissue; RVD±ELP-VEGF 13.4±3.2 pg/mg tissue, p<0.05), andattenuated the expression of pro-fibrotic transforming growth factor(TGF)-β, smad-4, and tissue inhibitor of matrix-metalloproteinase(TIMP)-1, whereas improved smad-7 and matrix-metalloproteinase (MMP)-2compared to untreated RVD, suggesting a potential decrease inpro-inflammatory, pro-fibrotic, and tissue remodeling activity (FIG. 12,top). Furthermore, ELP-VEGF therapy improved glomerular expression ofpodocin (FIG. 12, middle) and reduced nephrinuria, suggesting protectionof podocytes. Glomerulosclerosis and tubule-interstitial fibrosis(7.3±0.01 and 9.3±0.04%, respectively, p<0.05 vs. Normal) weresignificantly reduced (2.3±0.04 and 3.4±0.1%, respectively, p<0.05 vs.RVD and Normal) after ELP-VEGF therapy (FIG. 12, bottom). Similarly, MVmedia-to-lumen ratio (0.34±0.01, p<0.05 vs. Normal) was improved afterELP-VEGF therapy (0.18±0.05, p<0.05 vs. RVD, p=NS vs. Normal),suggesting attenuated MV remodeling in addition to the improvements inMV rarefaction.

This study supports the feasibility of ELP-VEGF therapy and suggeststherapeutic effects of this intervention.

Single-dose systemically-delivered ELP-VEGF improves renal function in aswine model of chronic RVD. Since ELPs have high affinity for renaltissue, preliminary studies were performed to determine whether asystemic administration may protect the kidney and improve renalfunction. To test this, 4 pigs with RVD were observed for 6 weeks,stenotic kidney function quantified, and then 2 of them treated with anintra-venous (IV) injection of ELP-VEGF via an ear vein cannula. Animalswere observed for 4 additional weeks, and renal function wasre-evaluated, observing that RBF and GFR in the stenotic kidney oftreated pigs were improved by over 70% compared to pre-treatmentfunction, as cortical MV rarefaction diminished (FIG. 13). These datasuggested renoprotective effects of ELP-VEGF even using a systemicroute.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list.

REFERENCES

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We claim:
 1. A pharmaceutical composition, comprising: a therapeuticallyeffective amount of elastin-like polypeptide (ELP) coupled to atherapeutic agent; a drug binding domain coupled to the ELP; and apharmaceutically acceptable carrier; wherein the ELP includes an aminoacid sequence having at least 5 repeats of the amino acid sequence GVPGX(SEQ ID NO: 1); wherein X in the sequence GVPGX (SEQ ID NO: 1) is anyamino acid except proline; wherein the drug binding domain does not bindthe therapeutic agent and wherein the therapeutically effective amountof the ELP coupled to the therapeutic agent improves the deposition andretention of the therapeutic agent in the kidney relative to anon-conjugated therapeutic.
 2. The pharmaceutical composition of claim1, wherein the therapeutic agent is VEGF, HGF, b-FGF, TGF-β, and HIF. 3.The pharmaceutical composition of claim 1, wherein the pharmaceuticallyacceptable carrier is for delivery intra-renally, intravenously,intraperitoneally, orally, intranasally, or subcutaneously.
 4. Thepharmaceutical composition of claim 1, further comprising a kidneytargeting agent coupled to the ELP, wherein the pharmaceuticalcomposition enhances the deposition and retention of the therapeuticagent in the kidney relative to the non-conjugated therapeutic.
 5. Thepharmaceutical composition of claim 1, wherein the ELP includes an aminoacid sequence comprising 5 repeats to 320 repeats of the amino acidsequence GVPGX (SEQ ID NO: 1).
 6. The pharmaceutical composition ofclaim 5, wherein the X in the amino acid sequence GVPGX (SEQ ID NO: 1)is Val, Ala, and Gly in a ratio range of 0-1:0-8:0-8.
 7. Thepharmaceutical composition of claim 6, wherein X is selected from Val,Ala, and Gly in a 1:8:7 ratio or 1:4:3 ratio, repeated between 5 and 320times.
 8. The pharmaceutical composition of claim 1, wherein the ELP isselected from amino acid sequences of SEQ ID NOs: 2, 3 and
 4. 9. Thepharmaceutical composition of claim 4, wherein the kidney targetingagent is selected from amino acid sequences of SEQ ID NOs: 5, 6, 7, and8.
 10. The pharmaceutical composition of claim 1, wherein the drugbinding domain is selected from amino acid sequences of SEQ ID NOs: 9,10, 11, and
 12. 11. The pharmaceutical composition of claim 2, whereinthe therapeutic agent is VEGF, and selected from VEGF₁₂₁, VEGF₁₆₅,VEGF₁₈₉, VEGF₂₀₆, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGF. 12.The pharmaceutical composition of claim 1, wherein at least one elementof the composition is coupled via a peptide, a non-cleavable modifiedpeptide bond, a protease cleavable peptide linker, or an acid labilechemical linker.
 13. A method of treating kidney disease in a subject inneed thereof, comprising: administering an effective amount of apharmaceutical composition, comprising an elastin-like polypeptide (ELP)coupled to a therapeutic agent, a drug binding domain coupled to theELP, and a pharmaceutically acceptable carrier; wherein the ELP includesan amino acid sequence having at least 5 repeats of the amino acidsequence GVPGX (SEQ ID NO: 1); wherein X in the sequence GVPGX (SEQ IDNO: 1) is any amino acid except proline; and wherein the drug bindingdomain does not bind the therapeutic agent.
 14. The method of claim 13,wherein said administration is intra-renally, intravenously,intraperitoneally, orally, intranasally, or subcutaneously.
 15. Themethod of claim 13, wherein the ELP includes an amino acid sequencecomprising 5 repeats to 320 repeats of the amino acid sequence GVPGX(SEQ ID NO: 1).
 16. The method of claim 13, wherein the X in the aminoacid sequence GVPGX (SEQ ID NO: 1) is chosen from: Val, Ala, and Gly ina ratio range of 0-1:0-8:0-8; or Val, Ala, and Gly in a 1:8:7 ratio or1:4:3 ratio, repeated between 5 and 320 times.
 17. The method of claim13, wherein the therapeutic agent comprises at least one growth factorselected from the group consisting of VEGF, HGF, b-FGF, TGF-β, and HIF.18. The method of claim 17, wherein the therapeutic agent is VEGF. 19.The method of claim 18, wherein the VEGF is selected from VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, andPlGF.
 20. The method of claim 13, wherein the kidney disease is selectedfrom a group consisting of chronic kidney disease, chronic renalvascular disease, chronic renal vascular disease caused by renal arterystenosis, and end-stage renal disease.
 21. The method of claim 13,wherein the pharmaceutical composition further comprises a kidneytargeting agent (KTA) coupled to the ELP, and wherein the KTA coupled tothe ELP of the pharmaceutical composition enhances the deposition andretention of the therapeutic agent in the kidney relative to apharmaceutical composition without the KTA.
 22. The pharmaceuticalcomposition of claim 2, wherein the therapeutically effective amount ofthe ELP coupled to the therapeutic agent treats kidney disease in asubject.
 23. The pharmaceutical composition of claim 22, wherein thekidney disease is selected from the group consisting of chronic kidneydisease, chronic renal vascular disease, and end-stage renal disease.24. A pharmaceutical composition, comprising: a therapeuticallyeffective amount of elastin-like polypeptide (ELP) coupled to atherapeutic agent, and a pharmaceutically acceptable carrier; whereinthe ELP includes an amino acid sequence having at least 5 repeats of theamino acid sequence GVPGX (SEQ ID NO: 1); wherein X in the sequenceGVPGX (SEQ ID NO: 1) is any amino acid except proline; wherein the ELPis selected from amino acid sequences of SEQ ID NOs: 2, 3 and 4; andwherein the therapeutically effective amount of the ELP coupled to thetherapeutic agent improves the deposition and retention of thetherapeutic agent in the kidney relative to a non-conjugatedtherapeutic.